Apurinic/Apyrimidinic (AP), or abasic, sites arise spontaneously in DNA with a calculated rate up to 10,000 bases per human cell per day. AP sites are cytotoxic and mutagenic and need to be repaired quickly in order to maintain the functional and genetic integrity of the genome. One of the major sources of AP sites is inherent instability of the glycosylic bond, found predominantly in purines. Abasic sites can also arise either by the actions of reactive oxygen species, or by enzymatic excision of damaged bases via the cleavage of the N-glycosyl bond catalyzed by a DNA glycosylase (See: Prokaryotic Base Excision Repair, Wilson III, D. M., Engelward, B. P. and Samson, L. (1998) pp.29-64; from: DNA Damage and Repair, V.1: DNA Repair in Prokaryotes and Lower Eukaryotes, Edited by: J. A. Nickoloff and M. F. Hoekstra, humana Press Inc., Totowa, N.J.).
AP sites in double-stranded DNA are recognized by a class of enzymes termed Class II AP endonucleases that cleave the phosphodiester backbone on the 5′ side of the AP site via a hydrolytic mechanism, thereby providing a free 3′-OH group that serves as a substrate for DNA polymerases to initiate Base Excision Repair (BER). The Endonuclease IV from Escherichia coli is one example of a Class II AP endonuclease. See: Regulation of Endonuclease IV as Part of an Oxidative Stress Response in Escherichia coli, Weiss B. (1998) pp.85-96; from: DNA Damage and Repair, V.1: DNA Repair in Prokaryotes and Lower Eukaryotes, Edited by: J. A. Nickoloff and M. F. Hoekstra, humana Press Inc., Totowa, N.J.
A number of DNA glycosylases that are called Class I AP endonucleases exhibit AP site-cleavage activity as part of their mechanism of action. However, these enzymes act as β-elimination catalysts, cleaving the phosphodiester backbone 3′ to the AP site, resulting in atypical 3′-termini, such as 3′-phosphoglycolate and 3′-phosphate. These atypical termini block the 3′-OH group that serve as a substrate for polymerases and are subject to subsequent repair by the Class II AP endonucleases that cleave the blocks and initiate the BER. See: Prokaryotic Base Excision Repair, Wilson III, D. M., Engelward, B. P. and Samson, L. (1998) pp.29-64; from: DNA Damage and Repair, V.1: DNA Repair in Prokaryotes and Lower Eukaryotes, Edited by: J. A. Nickoloff and M. F. Hoekstra, humana Press Inc., Totowa, N.J.; and Abasic Site Repair in Higher Eukaryotes, Strauss, P. R. and O'Regan, N. E. (2001) pp.43-86; from: DNA Damage and Repair, V.3: Advances from Phage to Human, Edited by: J. A. Nickoloff and M. F. Hoekstra, humana Press Inc., Totowa, N.J.
Polynucleotide identification assays that are based on a selective cleavage of a probe hybridized to a target nucleic acid have been disclosed by others. For example, U.S. Pat. Nos. 4,876,187; 5,011,769; 5,660,988; 5,731,146; 5,747,255 and 6,274,316 disclose nucleic acid probes having a scissile linkage incorporated as part of the nucleic acid backbone and in the middle of the nucleic acid probe. U.S. Pat. No. 5,403,711 also discloses a similarly designed DNA-RNA-DNA probe, wherein the embedded RNA sequence is a substrate for RNase H when duplexed. Hybridized probes with an incorporated cleavable linkage within the middle of the probe have a diminished duplex stability after the enzymatic cleavage. Their cleavable sites also are not exquisitely specific.
U.S. Pat. Nos. 5,516,663 and 5,792,607 disclose using endonuclease IV to remove an abasic site incorporated as a blocking agent on the 3′ end of an oligonucleotide to improve specificity and sensitivity of the ligase chain reaction (LCR) or polymerase chain reaction (PCR) amplification.
U.S. Pat. Nos. 5,656,430; 5,763,178; 6,340,566 disclose methods for detecting point mutations by using an endonuclease to cleave the nucleic acid backbone in the middle of the oligonucleotide at the point of mutation. In methods that identify a mismatch by enzymatic cleavage of a nucleic acid backbone, the presence, rather than the absence, of a mismatch stimulates the cleavage of the probe phosphodiester backbone.
U.S. Pat. No.6,309,838 discloses using labeled nucleotide excision repair enzymes to detect bound enzyme to DNA sequence impairments.
European Patent EP 1 071 811 B1 discloses a method of DNA synthesis from a 3′-OH generated by cleavage with a DNA glycosylase, but this method requires the steps of introducing a modified base and excising the modified base with a glycosylase followed with a treatment by AP endonuclease before carrying out the extension.
What is needed in the art is an assay which combines the advantages of target nucleic acid cycling, retained binding stability of the probe, an exquisitely specific cleavage site, the possibility for essentially instantaneous and highly sensitive reporter detection and the ability to directly combine detection with amplification procedures. Accordingly, there remains a need for compositions and methods that enable efficient detection of target nucleic acids with exquisite specificity. The present invention fulfills this need and others.